Initial report of a cutaneous T-cell lymphoma appearing on the auricular helix.

We report a case of cutaneous T-cell lymphoma in which the first sign of disease was involvement of the superior auricular helix. A review of the literature reveals that T-cell lymphoma often presents cutaneously, but it usually does not involve the ear, and an auricular lesion is rarely the first sign. The uncommon nature of this presentation, in addition to the potential need for multiple biopsies for a tissue diagnosis, can lead to a delay in diagnosis. When evaluating skin lesions in the head and neck, a high index of suspicion for cutaneous T-cell lymphoma is a key factor in its early diagnosis.

Trisomy 15, sex chromosome loss, and hematological malignancy.

We report 6 patients with myelodysplasia who, on routine cytogenetic studies, demonstrated trisomy 15. Four of these also had sex chromosome loss. A review of the literature revealed 6 other cases of trisomy 15 with sex chromosome loss and 22 cases of trisomy 15 as the sole chromosomal abnormality. All cases had hematologic malignancy or myelodysplasia. Trisomy 15 is uncommon but tends to be associated with myelodysplasia in older subjects, and with sex chromosome loss in about one third of cases.

Ultrasonographically guided direct gene transfer in utero: successful induction of beta-galactosidase in a rabbit model.

OBJECTIVE: We sought to determine whether the transfer of enzyme-encoding genes in utero can be detected after birth. STUDY DESIGN: An adenoviral vector carrying the gene for beta-galactosidase was injected under ultrasonographic guidance into the livers of 4 rabbit fetuses per litter (3 litters total) at 27 days' gestation. On delivery of the pups 2 to 3 days later, the livers were analyzed for beta-galactosidase activity by using 5-bromo-4-chloro-3-indolyl-beta-D -galactopyranoside (X-gal) staining. Polymerase chain reaction was also performed on liver extracts as an additional independent measure of successful vector delivery. RESULTS: Successful targeting of the livers of fetal rabbits was demonstrated by beta-galactosidase activity in the nuclei of liver serosal cells, parenchymal hepatocytes, or columnar cells of the gallbladder in 7 (58%) of 12 injected pups and by polymerase chain reaction in liver extracts from 10 (83%) of 12 injected pups. CONCLUSIONS: These results suggest that vectors that carry genes for specific enzymes can be delivered to fetal organs in utero and that expression of the enzyme can be detected after delivery.

Permanent rescue of lesioned neonatal motoneurons and enhanced axonal regeneration by adenovirus-mediated expression of glial cell-line-derived neurotrophic factor.

Axotomy of peripheral nerves in neonatal rats induces motoneuron death that can be delayed but not arrested by the application of several neurotrophic factors (NFs) or adenoviral vectors carrying genes for NFs. We tested whether an adenoviral vector carrying the gene for glial cell-line-derived neurotrophic factor (Adv.RSV-GDNF) would prevent neonatal motoneuron death after facial nerve transection or crush. Nerve transection eliminates the pathway for axonal regeneration, while nerve crush preserves the pathway necessary for target reinnervation that may be required for the permanent rescue of motoneurons. Both types of injury cause substantial motoneuron death in neonatal animals. Adv.RSV-GDNF or a control vector carrying the beta-galactosidase gene (Adv.RSV-betagal) was injected into facial muscles 2 days before the nerve was transected, or Adv.RSV-GDNF, Adv.RSV-betagal, Adv.d1312 (a vector lacking a transgene), or vehicle was injected into facial muscles immediately after nerve crush. Four weeks after nerve transection, few motoneurons survived after Adv.RSV-GDNF and Adv.RSV-betagal treatment (6.1% and 2.4%, respectively). Four weeks after nerve crush, 40% of the motoneurons survived after Adv.RSV-GDNF treatment but only 17% survived in control groups. By 20 weeks, 39% of the motoneurons of the Adv.RSV-GDNF treatment groups survived but only 15-19% survived in controls. The numbers of myelinated axons of the buccal nerve branch of Adv.RSV-GDNF treatment groups were also higher than controls at 4 and 20 weeks (24% and 100% compared to 4.4-6.2% and 25-33% for Adv.RSV-GDNF and controls, respectively). By 20 weeks, Adv.RSV-GDNF-treated animals recovered 50% of the contralateral vibrissal function, while in controls only 5-11% of function was restored.

Neuroprotection of spinal motoneurons following targeted transduction with an adenoviral vector carrying the gene for glial cell line-derived neurotrophic factor.

Application of neurotrophic factors (NFs) to the cut stump of peripheral nerves confers transient (1- to 2-week) neuroprotection of motoneurons from axotomy-induced death in neonates. We tested whether lumbar spinal motoneurons would be protected from axotomy-induced death when they were genetically modified to produce NFs in situ. Adenoviral (Adv) vectors carrying neurotrophic factor genes under control of the Rous sarcoma virus long terminal repeat promoter (Adv.RSV-nf) or a control vector containing the beta-galactosidase (beta-gal) gene (Adv.RSV-betagal) was injected into the hindlimb muscles of neonatal rats. The Adv were taken up by peripheral nerves and transported to lumbar spinal cord motoneurons where the transgenes were expressed. A fraction (18%) of the motoneurons that projected through the sciatic nerve were transduced with Adv.RSV-betagal. Expression of Adv.RSV-betagal was detected in motoneurons after 7 days and 3 weeks, with no evidence of vector- or beta-gal-induced toxicity or inflammation. PCR, immunocytochemistry, and RT-PCR demonstrated transport of the Adv.RSV-nf vectors to motoneurons and their expression. After retrograde transport of an Adv.RSV-nf vector carrying the gene for glial cell line-derived neurotrophic factor, a substantial proportion of the sciatic nerve motoneurons were resistant to axotomy-induced death 7 days and 3 weeks after sciatic nerve transection (56 and 44%, respectively), compared to Adv.RSV-betagal controls (2.5 and 0%, respectively).

Aberrant expression of GABAA receptor subunits in the tottering mouse: an animal model for absence seizures.

The single-locus mutant mouse tottering (tg) is an established model for absence seizures. We have previously reported an impairment in GABA-induced chloride uptake in tg brain [Tehrani and Barnes, Epilepsy Res. 1995;22:13-21]. In order to determine if this alteration in GABAA receptor function can be related to specific receptor isoforms, we examined the radioligand binding properties of GABAA receptors and the expression of GABAA receptor subunit mRNAs in the cerebral cortex. Saturation binding of [3H]flunitrazepam revealed a significantly lower Kd value in tg cortical tissues (1.77 +/- 0.05 nM) in comparison to that for the background C57BL/6J strain (3.23 +/- 0.23 nM), while the Bmax values were indistinguishable. Biphasic displacement of [3H]flunitrazepam binding by 2-oxoquazepam showed that low affinity binding sites account for 36 +/- 7.6 and 51 +/- 7.5% of the total in control and tg, respectively. The level of [35S]-t-butylbicyclophosphorothionate (TBPS) binding to tg cortical membranes was 73.6 +/- 5.8% of that in controls. Paired measurements by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) revealed no significant differences in the levels of GABAA receptor alpha 1, alpha 3, alpha 5, beta 2, beta 3, gamma 2 or gamma 3 subunit mRNAs between tg and control cortex. However, tg tissues showed elevated levels of alpha 2- and beta 1-subunit mRNAs, representing 256 and 177%, respectively, those of controls. For the tg cortex, the enhanced expression of GABAA receptor alpha 2 and beta 1 subunits correlates with recombinant subtypes known to have low affinity for 2-oxoquazepam and impaired binding of TBPS. These aberrant properties of GABAA receptors could influence the development or propagation of phenotypic seizures in the tottering mouse.

Targeted transduction of CNS neurons with adenoviral vectors carrying neurotrophic factor genes confers neuroprotection that exceeds the transduced population.

Application of neurotrophic factors (NFs) to the cut stump of motor nerves of neonatal rats confers neuroprotection from trauma-induced neuronal death. To test whether motoneurons are capable of responding to endogenously produced NFs, facial motoneurons were genetically modified in vivo to express several NFs and then tested for their response to peripheral nerve damage. Replication-defective adenoviral vectors [Adv. Rous sarcoma virus (RSV)-nf] representing three families of NFs were constructed that carried genes for brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), glial cell-derived neurotrophic factor (GDNF), and nerve growth factor. Media from cultured cells transduced with Adv. RSV-nf contained NFs that supported the survival of cultured chick sensory neurons in the same manner as recombinant NF standards. When Adv.RSV-nf or an adenoviral vector containing the beta-galactosidase gene (Adv.RSV-beta-gal) were injected into the facial muscles of neonatal rats the vectors were retrogradely transported to the facial nucleus where the NFs or beta-gal were expressed. A fraction (approximately 10%) of the neurons were transduced as demonstrated by reverse transcriptase-PCR, histochemistry, and immunocytochemistry. In the case of Adv.RSV-BDNF, Adv.RSV-CNTF, and Adv.RSV-GDNF, a significant portion of the facial nucleus neurons was protected, 16.5, 18.2, and 53.3%, respectively, from death after axotomy, showing that neurons are capable of transporting the Adv. RSV-nf, expressing the recombinant NF genes, and responding to the NFs. In the case of Adv.RSV-GDNF, a greater number of facial nucleus motoneurons survived than were transduced, indicating that neighboring untransduced neurons were protected by the GDNF expressed by the transduced neurons by a paracrine mechanism.

Clathrin-coated vesicles from bovine brain contain uncoupled GABAA receptors.

Clathrin-coated vesicles are thought to be a vehicle for the sequestration of GABAA receptors. For coated vesicles from bovine cerebrum, we examined the binding properties of [3H]muscimol. a GABAA-specific agonist. [3H]flunitrazepam a benzodiazepine agonist, and [35S]t-butylbiocyclophosphorthionate (TBPS), a ligand for GABAA receptor channels. Under standard conditions, the binding level of [3H]muscimol, [3H]flunitrazepam, and [35S]TBPS to coated vesicles represented 12.3 +/- 1.8%, 7.9 +/- 1%, and 10.2 +/- 1.8%, respectively, of that in crude synaptic membranes. Coated vesicles showed a single [3H]flunitrazepam binding site with a KD value (12 nM) which was 9-fold that for synaptic membranes. The allosteric coupling between binding sites was measured by the addition of GABA to [3H]flunitrazepam and [35S]TBPS binding assays. For [3H]flunitrazepam binding to synaptic membranes, GABA gave an EC50 = 2.0 microM and at saturation (100 microM) an enhancement of 122%. This stimulation was completely blocked by the GABA antagonist SR95531. In contrast, neither GABA nor SR95531 had a significant effect on [3H]flunitrazepam binding to CCVs, indicating that the allosteric interaction between GABA and benzodiazepine binding sites is abolished. Likewise, GABA displaced nearly all of the [35S]TBPS binding to synaptic membranes but had no effect on binding to coated vesicles, indicating that coupling between the GABA binding sites and chloride channel is also impaired. Thus GABAA receptors appear to be uncoupled during normal intracellular trafficking via coated vesicles. The presence of major GABAA receptor subunits on these particles was verified by quantitative immunoblotting. Relative to the levels in synaptic membranes, CCVs contained 110 +/- 14% and 29.5 +/- 3.8%, respectively, of the immunoreactivity for GABAA receptor beta 2 and alpha 1 subunits. Thus, in comparison to GABAA receptors on synaptic membranes, those on CCVs have a reduced alpha 1/beta 2-subunit ratio. It may be suggested that a selective decline in the content of alpha 1 subunits in coated vesicles could in part account for GABAA receptor uncoupling.

Agonist administration in ovo down-regulates cerebellar GABAA receptors in the chick embryo.

Chick embryos with an undeveloped blood-brain barrier were used to examine the down-regulation of GABAA receptors in vivo. The GABAA receptor agonist isoguvacine (5 mumol) was applied to the vascularized chorioallantoic membrane of 8 day embryos. This treatment was repeated on embryonic days 11, 14, and 17, and the embryos were sacrificed on day 18 (stage 42). Isoguvacine administration reduced the clonazepam-displaceable binding of [3H]flunitrazepam to washed cerebellar membranes by 34.0 +/- 3.0% compared to vehicle-treated controls. Binding reductions of lower magnitude were found in membranes from the cerebrum and optic lobes. Administration of isoguvacine had no significant effect on the wet weights of whole embryos or cerebella, the yield of cerebellar membranes, or the binding of [3H]N-methylscopolamine. The reduction of [3H]flunitrazepam binding to cerebellar membranes was dose-dependent, allowing a half saturation value of 8 microM isoguvacine to be estimated. Scatchard analysis showed that the Bmax for [3H]flunitrazepam binding was reduced by 28.3 +/- 6.7% compared to controls, without a change in the Kd. Embryonic exposure to isoguvacine also caused a reduction of 43.6 +/- 6.0% in the binding of the GABAA receptor channel ligand [35S]t-butylbicyclophosphorothionate to washed cerebellar membranes. Taken together, these results indicate that isoguvacine induces a down-regulation of the receptor subunits in vivo. However, measurements of cerebellar GABAA receptor mRNAs for the alpha 1, beta 2L, beta 2S, beta 4, gamma 1, gamma 2L, and gamma 2S subunits by reverse transcriptase-polymerase chain reaction (RT-PCR) revealed no significant alterations by isoguvacine administration. The data suggest that translational or post-translational mechanisms, rather than those modulating the synthesis or stability of subunit mRNAs, take precedence in establishing GABAA receptor down-regulation.

Developmental up-regulation and agonist-dependent down-regulation of GABAA receptor subunit mRNAs in chick cortical neurons.

We have used quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to analyze the expression of GABAA receptor subunit genes in cultured neurons from the chick embryo cerebral cortex. During maturation of the neurons between day 2 and day 8 in culture, levels of the alpha 1 subunit transcript (per ng total RNA) increased 3.8 +/- 0.3 fold, while those for the beta 2S and beta 4S subunits increased 2.4 +/- 0.4 and 1.8 +/- 0.2 fold, respectively. The accumulation of the beta 4 S subunit mRNA was more rapid than those encoding either the alpha 1 or beta 2S polypeptides. After 4 days in culture the beta 4S subunit transcript level reached 105 +/- 7.7% of that found after 8 days, while the corresponding amounts for the alpha 1 and beta 2S subunit mRNAs were 50 +/- 7.1% and 44 +/- 10.7%, respectively. On the other hand, no significant differences were observed in the level of either the gamma 1 or the gamma 2S subunit mRNA during development in vitro. Likewise, the ratios of the large/small splice variants (beta 2 = 0.16 +/- 0.02; beta 4 = 0.57 +/- 0.02; gamma 2 = 0.30 +/- 0.06) did not show detectable changes during this period. To study the down-regulation of the mRNAs, a single dose of 100 microM GABA was added to the culture medium. After 7 days of exposure to GABA, the levels of transcripts for the alpha 1, beta 2, beta 4, gamma 1, and gamma 2 subunits and their splice variants (where present) were all reduced by 47-65% compared to untreated controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Plastid Genes Encoding the Transcription/Translation Apparatus Are Differentially Transcribed Early in Barley (Hordeum vulgare) Chloroplast Development (Evidence for Selective Stabilization of psbA mRNA).

Chloroplast genomes encode rRNAs, tRNAs, and proteins involved in transcription, translation, and photosynthesis. The expression of 15 plastid genes representing each of these functions was quantitated during chloroplast development in barley (Hordeum vulgare). The transcription of all plastid genes increased during the initial phase of chloroplast development and then declined during chloroplast maturation. RNAs corresponding to rpoB- rpoC1-rpoC2, which encode subunits of a plastid RNA polymerase, and rps16, which encodes a ribosomal protein, reached maximal abundance early in chloroplast development prior to genes encoding subunits of the photosynthetic apparatus (rbcL, atpB, psaA, petB). Transcription of rpoB as well as 16S rRNA, trnfM-trnG, and trnK was high early in chloroplast development and declined 10-fold relative to rbcL transcription during chloroplast maturation. RNA hybridizing to psbA and psbD, genes encoding reaction center proteins of photosystem II, was differentially maintained in mature chloroplasts of illuminated barley. Differential accumulation of psbD mRNA relative to rbcL mRNA was due to light-stimulated transcription of psbD. In contrast, enhanced levels of psbA mRNA in mature chloroplasts were due primarily to selective stabilization of the psbA mRNA. These data document dynamic modulation of plastid gene transcription and mRNA stability during barley chloroplast development.

Quantitative analysis of transcription and RNA levels of 15 barley chloroplast genes. Transcription rates and mRNA levels vary over 300-fold; predicted mRNA stabilities vary 30-fold.

Higher plant plastid genomes encode rRNAs, tRNAs, and protein subunits of the RNA polymerase, ribosomes, and the photosynthetic apparatus which vary over 1000-fold in abundance. Quantitative analysis of transcription and RNA levels was carried out on 15 plastid genes which are located in 14 different transcription units covering 50% of the barley plastid genome. Transcription of 16S rRNA, trnfM-trnG, and trnK was high relative to most other plastid genes. Transcription of trnfM-trnG was 5 times greater than trnK indicating that differences in tRNA levels in plastids could be due, in part, to differences in transcription. Among the protein coding genes, mRNA levels varied over 900-fold and transcription over 300-fold. The gene showing the lowest transcription rate and mRNA level, rpoB, is located in a gene cluster which encodes subunits of the plastid RNA polymerase (rpoB-rpoC1-rpoC2). RpoA, which encodes the alpha subunit of the RNA polymerase, was located in a gene cluster encoding ribosomal proteins (rpl23, rps19, rpl16) and infA. RNA from this gene cluster is 30-fold more abundant than rpoB mRNA, suggesting that expression of rpoA is regulated at the level of translation or protein stability. Polycistronic operons encoding subunits of the photosynthetic apparatus (psbB-psbH-petB-petD; psbK-psbI-psbD-psbC; atpB-atpE; psaA-psaB) had higher transcription rates and correspondingly higher mRNA levels than genes which encode ribosomal proteins or RNA polymerase subunits. RbcL and psbA, which are located in separate transcription units, exhibited the highest transcription rates and mRNA levels. Correspondence between transcription rate, mRNA level, and protein abundance indicates that transcription is a primary determinant of barley plastid gene expression. In addition, a 30-fold variation in predicted mRNA stability was observed which further increases the dynamic range of plastid mRNA abundance.

Plastid DNA synthesis and nucleic acid-binding proteins in developing barley chloroplasts.

Activation of plastid DNA synthesis occurred early in barley leaf chloroplast development. High rates of DNA synthesis were observed in the leaf basal meristem of dark-grown plants where plastid transcription activity was low. DNA synthesis activity decreased in later stages of chloroplast development. Plastid nuclei acid-binding proteins were detected after lithium dodecylsulfate (LDS)-polyacrylamide gel separation and renaturation. One set of nucleic acid-binding proteins was associated with plastid nucleoids. A second set of nucleic acid-binding proteins co-sedimented with ribosomes and most probably corresponds to ribosomal proteins. Changes in the composition of the nucleic acid-binding proteins were characterized as a function of chloroplast development in dark-grown and illuminated barley plants.

Plastid transcription activity and DNA copy number increase early in barley chloroplast development.

Plastid transcription activity and DNA copy number were quantified during chloroplast development in the first foliage leaf in dark-grown and illuminated barley (Hordeum vulgare L.) seedlings. Primary foliage leaves of seedlings given continuous illumination from 2 days post-imbibition reached a final mean length of 15 centimeters at 6.5 days, whereas primary leaves of dark-grown seedlings required 7 days to reach a similar length. Dividing cells were observed in the basal 0.5 to 1 centimeter of primary leaves until 5.5 days post-imbibition. Plastids isolated from cells located in the basal meristem of 4-day-old seedlings were small ( approximately 2 micrometers in diameter), exhibited low transcription activity and contained approximately 130 copies of plastid DNA per organelle. Cell size increased from 18 to 60 micrometers in a 1 to 3 centimeter region located adjacent to the leaf basal meristem. In this region, transcriptional activity per plastid increased 10-fold and DNA copy number increased from 130 to 210. Plastid transcriptional activity declined rapidly in illuminated plants with increasing leaf cell age and plastid DNA copy number also declined but with a slower time course. In dark-grown seedlings, plastid transcriptional activity declined more slowly than in illuminated plants while DNA copy number remained constant with increasing cell age. These data show that plastid transcriptional activity and DNA copy number increase early in chloroplast development and that transcriptional activity per DNA template varies up to 5-fold during barley leaf biogenesis.